A total internal reflection fluorescence (TIRF) microscope uses an evanescent wave, which is generated during a total reflection of incident light, to detect a fluorescence signal in a vertical direction for a local area of a specimen dyed with fluorescent material, and to provide the corresponding image information. The TIRF microscope is often used for research in the fields of cell biology, molecular biology, and medicine, and in particular, is used directly in research concerning various protein reactions occurring at the surface of a cell or changes in the cell surface caused by a drug.
In the basic configuration of a conventional TIRF microscope, an evanescent wave localized in a depth direction, which is created when incident light is totally reflected off the interface between a specimen and the substrate, excites the fluorescent molecules with which the specimen is dyed, and afterwards the fluorescence signal emitted by the excited fluorescent molecules is detected and imaged. However, with the conventional TIRF microscope, it is impossible, or at least difficult, to detect molecules or movement paths of molecules that are smaller than the resolution limit in the horizontal direction calculated by Abbe's diffraction equation. Thus, there is a need for a TIRF microscope that has a high resolution not only in the depth direction but also in the horizontal direction.